In the early 1980s the hypothesis was advanced that cholesterol synthesis is regulated via an oxysterol binding protein, i.e., a cytosolic component that bound to oxysterols. Today two protein families have been described which are known to bind oxysterols: the liver X receptors (LXRs), and the protein family described as cytoplasmic oxysterol receptors, termed oxysterol binding proteins (OSBPs) or OSBP-related proteins (ORPs).
OSBP (oxysterol-binding protein) is a protein found in eukaryotes that was first identified based on its high affinity for oxysterols, especially 25-hydroxycholesterol (Kd=37 nM). It is the founding member of a family of evolutionarily conserved proteins; at least is twelve are now known in mammals, including OSBP (also referred to as OSBP1), ORP1L, ORP1S, ORP2, ORP3, ORP4L, ORP4S, ORP5, ORP6, ORP7, ORP8, ORP9L, ORP9S, ORP10, and ORP11. Lehto et al. (2003) Biochim Biophys Acta 1631:1-11; Yan et al. (2008) Int Rev Cytol 265:253-85; Fairn et al. (2008) Cell Mol Life Sci 65:228-36. OSBP1 is an 89 kD protein comprising a sterol-binding domain and several other domains involved in protein-protein and protein-lipid interactions. These domains include a pleckstrin homology (PH) domain, which localizes proteins to phosphatidylinositol-containing membranes, and an FFAT domain, which is an endoplasmic reticulum (ER)-localizing domain. OSBP1 does not exhibit enzymatic activity. Although an understanding of its cellular function remains incomplete, studies in the last few years have revealed that OSBP1 is a sterol sensor that can exert control over key signaling pathways.
In addition to OSBP1, mammals express at least eleven other OSBP-related proteins referred to as ORPs. Like OSBP1, each ORP contains a sterol-binding domain, although beyond that there is considerable variation in ORP configuration. Most ORPs include a PH domain in addition to the sterol-binding domain. The sterol-binding domain of OSBP/ORPs is conserved among the family of twelve proteins, but it is not similar to sterol-binding domains of other proteins such as LXR, Insig, or NPC-1. The ORPs are less well characterized than OSBP1, although they have been implicated in lipid metabolism, signaling, vesicular traffic, and non-vesicular sterol transport. Currently, their cellular functions are not well understood, although it is believed that they play important cellular roles since they are expressed ubiquitously and they are evolutionarily conserved. They have been implicated in atherosclerosis and possibly cancer. For instance, overexpression of ORP1L leads to atherosclerotic lesions in mice. Additionally, atherosclerotic lesions were found to contain up-regulated ORP8, and ORP9 has been identified as a therapeutic target in raising high-density lipoprotein (HDL) levels. Knockdown of OSBP1 or ORP8 led to enhanced levels of cholesterol efflux upon LXR agonism. Increased cholesterol efflux is a therapeutic approach in the treatment of atherosclerosis, suggesting that ORPs may be atherosclerosis drug targets. In yeast, there are 7 Osh proteins (ORP homologs). Interestingly, yeast can survive with any six of their seven Osh proteins deleted, but not all seven.
Cephalostatin 1 (1), OSW-1 (2), ritterazine B (3), schweinfurthin A (4), and (−)-stellettin E (5) (see FIG. 1) are natural products that are potently cytotoxic to selected human cancer cell lines with half-maximal growth inhibitory concentrations (GI50) in the low nanomolar range. These molecules also have highly similar cytotoxicity patterns against the sixty cultured human cancer cell lines evaluated by the National Cancer Institute (NCI-60), strongly suggesting that they share a cellular target or act by similar mechanisms. Compounds with Pearson correlation coefficients (p values) greater than 0.6 (1.0 is a perfect match) by COMPARE analysis are considered mechanistically related. These compounds previously have been referred to as CRAMs (Cephalostatin and Related Antiproliferative Molecules).
WO 2010/068877 discloses that CRAMs and certain synthetic analogs thereof are high affinity ligands of oxysterol binding proteins (OSBPs) and OSBP-related proteins (ORPs), including OSBP1 and ORP4.